Capillary electrophoresis (CE) is a chemistry separation technique which utilizes the differences in solute electrophoretic velocity to isolate the various components of a sample. Electro-osmotic flow is the bulk flow of buffer from a first buffer reservoir to a second buffer reservoir through the capillary due to the shearing movement of a diffuse layer of cations past a more firmly held, dense layer, interacting with integral, anionic groups of the capillary wall.
Factors which influence the velocity of electro-osmotic flow are: electrical field strength; buffer dielectric constant; zeta potential (the electrical potential existing between diffuse and compact cationic layers); and buffer viscosity (which is dependent on bulk properties of the buffer and the temperature of the buffer).
Electrophoretic force is the force applied to charged particles residing in an electrical field, and neutral or uncharged molecules are not affected. Positively charged molecules (cations) migrate towards the cathode while negatively charged molecules (anions) move towards the anode. Factors controlling solute electrophoretic velocity are: molecular charge; electrical field strength; viscosity of the migration media; and solute molecular geometric factors.
The net velocity at which a solute travels in an uncoated, open capillary tube during CE is the vector sum of the electro-osmotic and electrophoretic velocities. Buffer viscosity plays a significant role for both of these phenomenon. Both electrophoretic and electro-osmotic velocities are inversely proportional to buffer viscosity, thus affecting the net migration velocity for all solutes. When an electrical field is applied to a capillary which contains buffer, joule heating occurs.
Joule heating is a major problem in capillary electrophoresis (CE). At one extreme, the solution can be heated to boiling, putting an end to the separation. Even at temperatures below boiling, elevated internal temperature increases diffusional spreading and Taylor dispersion. (See A. Guttman el al., J. Chromatogr., 559:285-294 (1991); S. L. Petersen el al., Anal. Chem., 64:1676-1681 (1992); J. H. Knox, Chromalographia, 26:329-337 (1988).)
Some analytes, such as proteins, are themselves heat sensitive, and undergo irreversible changes at temperatures well below the boiling point of water. For such reasons, CE is usually performed in low ionic strength buffers or at moderate voltages (10-25 kV), or both.
Although heat dissipation from a capillary can be efficient, often it is not perfect. In typical buffers, temperature elevation in capillaries is readily observable at normal (100-350 V/cm) operating electric fields. (See K. D. Davis el al., Anal. Chem., 65:293-298 (993); K. L. Liu et al., Anal. Chem., 66:3744-3750 (1994).)
Several methods of capillary temperature control are presently employed, including forced air convective cooling and the use of liquid coolant. For example, U.S. Pat. No. 5,021,646 to Weinberger et al., hereby incorporated by reference, describes a capillary electrophoresis unit utilizing an air cooled cartridge. The temperature of the capillary is determined by measuring its electrical resistance. When the temperature of the capillary requires adjustment, a fan drives air cooled by a Peltier heat sink across the capillary in the cartridge. Similarly, U.S. Pat. No. 5,122,253 to Christianson, hereby incorporated by reference, describes the use of a stream of pressurized gas in a transverse flow through a capillary region. A rotary fan creates a gas flow which is axial to the helix formed by the capillary tube. This is intended to cool the capillary.
A liquid cooling apparatus is taught by U.S. Pat. No. 5,198,091 to Burolla et al., hereby incorporated by reference. The cooled capillary cartridge is bisectional and contains an inner chamber. The inner chamber holds the capillary and also can contain a circulating liquid coolant. The coolant is described as either water or, preferably, a completely fluorinated hydrocarbon. U.S. Pat. No. 5,164,064 to Dill et al., hereby incorporated by reference, reports an improved liquid cooled device. The device includes a capillary cartridge with a coolant flow channel. The coolant flow channel is advertised as having little or no dead volume to improve uniform cooling.
U.S. Pat. No. 5,183,101 to Penaluna et al., hereby incorporated by reference, combines a liquid cooled system with a refrigeration device. The device is comprised of a heat exchanger, a compressor and a capillary. The housing and electrical units of the refrigeration system are electrically insulated from the buffer solution, and the buffer solution is passed through the refrigeration coils to cool it as necessary. Alternatively, warm coolant from the compressor discharge can be directed to the heat exchanger for the warming of the electrode buffer as necessary.
All of the above-described approaches have shortcomings. For example, liquid cooling can be effective, but it is complicated. The capillary must be surrounded with a flowing, heat-conductive liquid; this presents concerns regarding leakage and electric shortages in the system. Forced air cooling is limited in its capacity to remove heat from the capillary column. This lack of an efficient system for removing heat from the capillary device limits the usefulness of capillary electrophoresis as a whole.
What is needed is a cooling means that will cool the capillary in an efficient manner to expand the usefulness of capillary electrophoresis.